Maya Sim - Week 6

   On Monday, I had to start the week with sacrificing mice that stopped showing signal in the encapsulation devices. When we imaged these mice with IVIS using luciferase, we saw nothing in the location where we implanted the device a month ago. Nino hypothesized that it was due to mice constantly moving and the device crumpling up in a ball as a result. Even if there is vascularization of the device, the cells inside the device would not be able to achieve effective nutrient and waste exchange, gradually leading to cell death. When we sacrificed these mice and opened up the surgical site, it was proven that Nino was correct – the flat shape of the device was now unrecognizable. Surrounded by a layer of fat tissue, the device became useless in providing the cells with necessary support.

   Fortunately, we recently received newly engineered encapsulation devices from a collaborating lab. The shape was almost identical except for the rigid ring outlining the device. We are hoping that this ring will provide the extra support and prevent the device from crumpling up. As the new device was less floppy and easier to handle with forceps, the pre-implantation procedure could be completed in much less time with no complications. After the pre-implantation of the device, we need to wait for 2 weeks so that it vascularizes. Although I will still be at the lab for the infusion of cells, I am disappointed that I will not be able to image the mice for the results.

   Throughout the week, I had various samples that I had to isolate mitochondria from. Starting with mice, I took their spleen, liver, and skeletal muscle. Using a metal mesh on a plate containing some lysis buffer, I homogenized the spleen and the liver. For the skeletal muscle, I had to use a scalpel to cut it into 1-3mm pieces. After transferring them into smaller tubes, I homogenized them further using a pestle, then spun it down in a centrifuge. I threw out the supernatant and resuspended the pellet. Then, I took 2 microliters of the solution containing cells and mixed it with trypan blue to dye the living cells. Pipetting the solution into the hemocytometer, I counted the living cells that was within the boundaries using the manual cell counter. All I had to do was press on the clicker with a finger every time I saw a cell. It seemed easy enough, so I refused Nino’s offer to help with one of the samples. That was a grave mistake – after counting cells for all three samples, I could no longer feel my pointer finger. After approximating the total cell count by multiplying the dilution factors, I followed the protocol from the week before to isolate the mitochondria. Then, I stained them with MitoTracker, ran the FACS analysis, and was relieved to find the yield of mitochondria higher than our previous attempt.

   The next day, I was once again busy with more samples given to me by Casey, a surgeon working with Nino. He had just gone to Lake Tahoe on a donor run and returned with fresh pieces of organs from an actual human donor. As part of an experiment to see if stressed islets would take in mitochondria, he asked me to isolate the mitochondria from the liver, thyroid, and spleen. Dumbfounded that he asked ME to handle human tissue, I just stared back at him with raised eyebrows. Apparently, Nino told him that I was a pro at isolating mitochondria.

   Feeling under high pressure, I was overly cautious in handling human tissue. I took forever to homogenize them, triple-checked the labels on each tube before I transferred anything over, and made sure I did not aspirate the pellet at all times. Although it took me an entire day to complete the isolation and run the FACS analysis, I impressed myself by the continuously improving yield of mitochondria (yay). These isolated mitochondria were then added to plates of human islets of Langerhans. These cells were kept in nutrition deprived media (1:100 dilution of replete media) for 24 hours. Using imaging tools, we would try to see if the stressed islets would take in the mitochondria from other tissues to improve their state.  

   While I am proud of myself for being recognized as “a pro” at isolating mitochondria, I also hope that I will be able to learn new skills next week.